This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The mechanisms by which ethanol causes liver cell injury are not clear. A major pathway that is a focus of considerable research in recent studies is the role of oxidative stress in alcohol toxicity. Induction of the 2E1 isoform of cytochrome P450 (CYP2E1) by ethanol appears to be one of the central mechanisms by which ethanol generates a state of oxidative stress. We have previously shown that damage to mitochondria by CYP2E1-derived oxidants is an early event in the overall pathway of cellular CYP2E1-dependent toxicity. However, the mechanistic link between CYP2E1 overexpression, ethanol metabolism and mitochondrial damage is currently unknown. The central hypothesis of this proposal is that CYP2E1-derived oxidants induce mitochondrial DNA (mtDNA) damage in liver cells exposed to ethanol, and this mtDNA damage causes mitochondrial dysfunction. We will test our central hypothesis by pursuing the following two specific aims: Specific Aim 1. To test whether mtDNA is a target of CYP2E1-derived oxidant species in vitro. We hypothesize that one consequence of CYP2E1 overexpression is mtDNA damage via increased generation of reactive oxygen species. Studies will be conducted using genetically engineered liver cells that overexpress CYP2E1, and control (parental) liver cells incubated with ethanol in vitro. We expect that CYP2E1 overexpressing liver cells will develop more mtDNA lesions evaluated with long distance qPCR than non-CYP2E1 expressing cells, and that mtDNA damage will be prevented by antioxidants. Specific Aim 2. To determine if CYP2E1 causes the mtDNA damage by ethanol in vivo. Genetically engineered mice (CYP2E1 knock-out) and wild type mice will be treated with ethanol (acute and chronic models). We hypothesize that CYP2E1 knock-out mice will be protected from ethanol-induced mtDNA damage. We expect that liver mtDNA isolated from alcohol-fed wild-type mice will show higher levels of damage than alcohol-fed CYP2E1-knockout mice.